Remodeling the zonula adherens in response to tension and the role of afadin in this response

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.     

Multiple locus genome-wide association studies for important economic traits of oil palm

Palm oil has a balanced fatty acid composition and has no trans fat. As a result, its use in food has increased as food-labeling laws have changed to specify trans fat content. Increasing oil production is the main goal in oil palm breeding. Genetic mapping and genomic studies in palm trees are necessary to understand the genetic architecture of economic traits of importance for palm oil production. To help achieve this, we sampled 422 oil palms from MPOB (Malaysian Palm Oil Board)­Angola germplasm collection and measured 13 economic traits from these palms. Multi-locus genome-wide association studies (GWAS) were conducted using least absolute shrinkage and selection operator (LASSO) and genome-wide efficient mixed model analysis. We identified 19 quantitative trait loci (QTLs) for 8 traits. Of these, four Angola-specific QTLs associated with bunch components were detected on chromosomes 4, 8, and 11. These QTLs are potentially useful for introgression of desirable genes from the Angola palms to advanced breeding populations for improvement of bunch and oil yield traits. The majority of the QTLs were detected by LASSO-A, in which the p values of individual markers were calculated based on bootstrapped standard errors. Many of the detected QTLs are nearby known QTLs detected from linkage studies reported by other research groups. We also conducted genomic selection (GS) for the 13 traits and concluded that GS can be an effective tool for oil palm breeding. This is the first GWAS and GS study conducted on oil palm germplasm from Angola, and the results can be very useful in oil palm genetic studies and breeding.     

The Juxtamembrane Region of the Cadherin Cytoplasmic Tail Supports Lateral Clustering, Adhesive Strengthening, and Interaction with p120ctn

Cadherin cell–cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308–315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94–amino acid region of the cytoplasmic tail, but not the β-catenin–binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120^ctn. These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120^ctn is involved in clustering and cell adhesion.     

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.     

A shortened dengue IgM capture ELISA using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody

A shortened IgM capture ELISA for the detection of dengue IgM antibodies using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody was described. The shortened two-step assay was compared with the four-step IgM capture ELISA on sera from dengue patients confirmed by the hemagglutination inhibition (HI) test. When paired acute and convalescent sera were tested, the shortened ELISA showed 100% agreement with HI results. It detected dengue IgM antibodies in the acute sera of 66% of patients with a primary dengue infection, 60% of patients with a secondary infection, and 98% of patients with a presumptive secondary infection. When the results of 151 dengue patients were combined, 75% of the acute sera were positive by the shortened IgM capture ELISA.     

Manipulation of Cell:Cell Contacts and Mesoderm Suppressing Activity Direct Lineage Choice from Pluripotent Primitive Ectoderm-Like Cells in Culture

In the mammal, the pluripotent cells of embryo differentiate and commit to either the mesoderm/endoderm lineages or the ectoderm lineage during gastrulation. In culture, the ability to direct lineage choice from pluripotent cells into the mesoderm/endoderm or ectoderm lineages will enable the development of technologies for the formation of highly enriched or homogenous populations of cells. Here we show that manipulation of cell:cell contact and a mesoderm suppressing activity in culture affects the outcome of pluripotent cell differentiation and when both variables are manipulated appropriately they can direct differentiation to either the mesoderm or ectoderm lineage. The disruption of cell:cell contacts and removal of a mesoderm suppressor activity results in the differentiation of pluripotent, primitive ectoderm-like cells to the mesoderm lineage, while maintenance of cell:cell contacts and inclusion, within the culture medium, of a mesoderm suppressing activity results in the formation of near homogenous populations of ectoderm. Understanding the contribution of these variables in lineage choice provides a framework for the development of directed differentiation protocols that result in the formation of specific cell populations from pluripotent cells in culture.     

Maximizing interferon-gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling

The Chinese hamster ovary (CHO) cell line producing interferon-gamma (IFN-gamma) exhibits a 2-fold increase in specific productivity when grown at 32 degrees C compared to 37 degrees C. Low temperature also causes growth arrest, meaning that the cell density is significantly lower at 32 degrees C, nutrients are consumed at a slower rate and the batch culture can be run for a longer period of time prior to the onset of cell death. At the end of the batch, product concentration is doubled at the low temperature. However, the batch time is nearly doubled as well, and this causes volumetric productivity to only marginally improve by using low temperature. One approach to alleviate the problem of slow growth at low temperature is to utilize a biphasic process, wherein cells are cultured at 37 degrees C for a period of time in order to obtain reasonably high cell density and then the temperature is shifted to 32 degrees C to achieve high specific productivity. Using this approach, it is hypothesized that IFN-gamma volumetric productivity would be maximized. We developed and validated a model for predicting the optimal point in time at which to shift the culture temperature from 37 degrees C to 32 degrees C. It was found that by shifting the temperature after 3 days of growth, the IFN-gamma volumetric productivity is increased by 40% compared to growth and production at 32 degrees C and by 90% compared to 37 degrees C, without any decrease in total production relative to culturing at 32 degrees C alone. The modeling framework presented here is applicable for optimizing controlled proliferation processes in general.     

A ferrocene-perfluoroarene molecular complex

Perfluorophenanthrene and decamethylferrocene cocrystallize as a molecular adduct in monoclinic space group P2₁/c with a = 8.842(2), b = 11.262(1), c = 30.695(8) Å, β = 95.89(2)°, V = 3040.3(8) ų, Z = 4. The structure was refined to R = 0.0537 for 1567 observed reflections. The perfluoroarene is twisted and chiral; the crystal is a racemate, however.